In the developing brainstem and spinal cord, distinct classes of motor neurons and ventral interneurons are generated by the graded signaling activity of the secreted protein Sonic Hedgehog (Shh). Shh controls neuronal fate by establishing different progenitor cell populations in the ventral neural tube that are defined by the expression of the transcription factors Pax6 and Nkx2.2. Pax6 functions as a key intermediary in the Shh-mediated control of motor neuron subtype identity. In the caudal hindbrain, elimination of Pax6 expression alters motor neuron subtype identity, transforming hypoglossal to vagal motor neurons. The mechanism by which Pax6 functions to control ventral neuronal fate remains unclear. Shh inhibits the expression of Pax6 in a concentration-dependent manner such that an inverse gradient of Pax6 results from the expression of secreted Shh by ventral midline structures. The specific aims of this proposal include determining whether the graded expression of Pax6 is important for the control of neuronal fate and not simply an insignificant consequence of the requirement for graded Shh signaling. Pax6 is expressed as two alternatively spliced proteins with distinct DNA binding characteristics. The regulation of this alternative splice choice by graded Shh signaling will be investigated as a possible mechanism by which graded Shh signaling generates cellular diversity within the spinal cord. Finally, a PCR-based cloning strategy will be used to differentially screen cDNA libraries constructed from single ventral progenitor cells specific to a particular motor neuron subtype to isolate genes involved in the specification of motor neuron subtype identity. The role of these genes in motor neuron development will be examined by studying the effect of their ectopic expression on motor neuron identity in the developing spinal cord. Through a more complete understanding of the genetic events which control motor neuron identity and early differentiation in the developing central nervous system, these experiments will provide considerable insight into the pathogenesis of amyotrophic lateral sclerosis (ALS) and other motor neuron diseases which result from the selective degeneration of this neuronal population. The manipulation of the developmental programs which control the specification of motor neurons may make possible novel therapeutic strategies to restore this specific subclass of central neurons lost either by traumatic spinal cord injury or progressive neurodegenerative disease.